Back to EveryPatent.com
| United States Patent |
5,017,375
|
|
Appel
, et al.
|
May 21, 1991
|
Method to prepare a neurotrophic composition
Abstract
The present invention is based on the discovery that amyotrophic lateral
sclerosis (ALS), Parkinson disease and Alzheimer disease are due to lack
of a disorder-specific neurotrophic hormone or factor. Diagnosis is
accomplished by assaying factors specific for a particular neuronal
network or system; for example, dopamine neutotrophic hormones from
striatum or caudate-putamen in the nigrostriatal dopaminergic neural
system are used to diagnose and treat parkinsonism. With tissue culture,
the presence or absence of spacific neurotrophic factos can be assessed in
ALS, parkinsonism, and Alzheimer disease. If there is a deficiency,
extracted and purified neurotrophic factors specific to the particular
neuronal network or system can be injected into a patient having ALS,
Alzheimer disease or parkinsonism for treatment of the disease.
| Inventors:
|
Appel; Stanley H. (Houston, TX);
Tomozawa; Yasuko (Houston, TX)
|
| Assignee:
|
Baylor College of Medicine (Houston, TX)
|
| Appl. No.:
|
052087 |
| Filed:
|
May 18, 1987 |
| Current U.S. Class: |
424/570; 435/4; 435/70.3; 514/21; 530/300; 530/412; 530/416; 530/417; 530/427; 530/839 |
| Intern'l Class: |
A61K 037/02; A61K 035/34 |
| Field of Search: |
424/95
435/68,4
514/21
530/300,412,416,417,427,839
|
References Cited
U.S. Patent Documents
| 3989819 | Nov., 1976 | Brockman | 424/95.
|
| Foreign Patent Documents |
| 0082612 | Nov., 1982 | EP.
| |
| 2420544 | Nov., 1979 | FR | 424/95.
|
| 73526 | Apr., 1984 | JP | 530/839.
|
Other References
Appel, Ann Neurol, 1981, 10:499-505.
Tomozawa and Appel, Society for Neuroscience Abstracts, No. 313.10, vol.
II, Part 1; 15th Annual Meeting; Dallas, Texas 10/20/85.
Appel et al; Tropic Factors and Degenerative Neuroligic Disease, May 19-20,
1986, International Conference on Neuroplasticity, Madrid, Spain.
Tomozawa and Appel, Brain Research, 1986, 399:111-124.
|
Primary Examiner: Stone; Jacqueline M.
Attorney, Agent or Firm: Davis Hoxie Faithfull & Hapgood
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a continuation-in-part of U.S. Ser. No. 768,887,
filed 23 Aug. 1985, now abandoned, which is a divisional of U.S. Ser. No.
444,293, filed 24 Nov. 1982, now abandoned.
Claims
We claim:
1. A method to prepare a neurotrophic composition, which method comprises:
(a) extracting factors from caudate-putamen tissue of a normal mammal;
(b) assaying the caudate-putamen extract for trophic effects on
dopaminergic neurons obtained from substantia nigra as measured by the
ability to stimulate dopamine uptake by said neurons; and
(c) isolating factors having said trophic effects.
2. The method of claim 1 wherein said normal mammal is of bovine source.
3. The method of claim 1 wherein said extraction comprises homogenizing the
tissue extract in aqueous solution, centrifuging the homogenate, and
recovering the supernatant.
4. The method of claim 1 which further comprises purifying the isolated
factors by fractionation.
5. The method of claim 4 wherein said purification yields dopaminergic
neurotrophic factors having molecular weights less than 3,000 daltons.
6. The method of claim 5 wherein a dopaminergic neurotrophic factor has a
molecular weight range of about 1,000 to about 1,600 daltons.
7. The method of claim 5 wherein a dopaminergic neurotrophic factor has a
molecular weight range of about 1,500 to about 1,800 daltons.
8. The method of claim 5 which further comprises purifying the dopaminergic
neurotrophic factor by cation-exchange HPLC chromatography.
9. The method of claim 3 wherein said tissue extract is prepared from fresh
tissue and said aqueous solution comprises a buffer supplemented with one
or more protease inhibitors.
10. The method of claim 9 which further comprises purifying said isolated
factors by gel filtration chromatography.
11. The method of claim 10 wherein said purification yields dopaminergic
neurotrophic factors having molecular weights less than 3,000 daltons.
12. The method of claim 5 wherein a dopaminergic neurotrophic factor has a
molecular weight range of about 1,800 to about 2,600 daltons.
13. The method of claim 11 wherein a dopaminergic neurotrophic factor has a
molecular weight range of about 1,500 to about 1,800 daltons.
14. The method of claim 11 wherein a dopaminergic neurotrophic factor has a
molecular weight range of about 1,000 to about 1,600 daltons.
15. The method of claim 11 wherein said purification further comprises
purifying the dopaminergic neurotrophic factors by anion-exchange HPLC
chromatography.
16. A factor prepared by the method of claim 7.
17. A factor prepared by the method of claim 12.
Description
FIELD OF THE INVENTION
The field of the invention is the diagnosis and treatment of ALS.
Parkinson's disease, and Alzheimer disease by neurotrophic factors.
BACKGROUND OF THE INVENTION
The causes of some of the most common and most devastating diseases of the
nervous system remain unknown. Prominent on this list are amyotrophic
lateral sclerosis (ALS), parkinsonism, and Alzheimer disease. Each of
these conditions is presently considered to be a degenerative disorder of
unknown origin. In each, viral or immunological causes have been
suggested, but no convincing reproducible data support the presence of an
infectious agent or a cell-mediated or humoral immune factor. All three
diseases reflect pathological change in a relatively limited network
within the peripheral or central nervous system, or both.
Amyotrophic Lateral Sclerosis
Amyotrophic lateral sclerosis is the name given to a complex of disorders
the compromise upper and lower motor neurons. Patients may present with
progressive spinal muscular atrophy, progressive bulbar palsy, primary
material sclerosis, or a combination of the conditions. The majority of
patients have components of all three types, but each form may represent
the sole clinical manifestation of motor system involvement [1].
(Reference numbers are to references listed at the conclusion of the
"Background of the Invention"). At the present time in the United States,
the incidence of the combined disease is approximately 1.8 per 100,000 [2]
and its prevalence is between 5 and 7 per 100,000. Males are affected more
commonly than females, the ratio of males to females being 1.6:1.
Approximately 10% of the cases are familial [3]. Onset may occur at any
age but is most common in the later decades, and the incidence appears to
increase with age. The mean age of onset is 66 years [6].
Distal weakness and atrophy are the hallmarks of the disorder, and both
upper and lower motor neurons are affected. Sensory signs are usually
absent. although quantitative sensory assessment by electromyography may
indicate abnormalities [4]. The extraocular muscles and bladder are rarely
involved. Progression usually occurs over 12 to 30 months, and death
ensues as a result of severe impairment of breathing functions.
The major pathological abnormality is loss of large motor neurons of the
motor cortex, brain stem, and spinal cord. In the remaining motor neurons
there is chromatolysis and inclusions that are rich in ribonucleic acid or
are Lewy body-like or eosinophilic (Bunina bodies) [5]. The whole neuron
seems to be involved, and there is only minimal evidence of "dying back"
of the peripheral axons [6]. In addition, large proximal axonal swellings
(spheroids) have been reported in motor neurons from patients with ALS
[7], and similar abnormalities can be induced in animals following
injection of B-B'-iminodiproprionitrile, with resulting impairment of slow
axonal transport [8]. These spheroids represent abnormalities of
neurofilaments and may be found in the cytoplasm as well as in the axon.
Involvement of the motor system has been described in familial conditions
appearing at earlier ages [3]. For example. Werdnig-Hoffman disease
present in utero or in infancy as a rapidly progressive autosomal
recessive condition characterized by severe weakness. Kugelberg-Welander
disease is first seen in the juvenile period with weakness in the hips and
subsequent involvement of the shoulder muscles. It is also inherited as an
autosomal recessive disorder, although autosomal dominant and X-linked
recessive transmission have been described. Both of these clinical
conditions result from anterior horn cell abnormalities and share clinical
features with the progressive muscular atrophies appearing later in life.
Parkinsonism
The presence of tremor, bradykinesia, and rigidity and loss of postural
reflexes are characteristics of idiopathic parkinsonism. At the present
time in the United States, the incidence of this disorder is estimated to
be approximately 20 per 100,000, and its prevalence is 200 per 100,000
[9]. There is a slight male-to-female preponderance with the ratio of
males to females being 1.2:1. The mean age of onset appears to be greater
than 67 years and, as in ALS, the incidence may increase with age. As in
ALS, some 5 to 10% of patients have a family history of the disorder.
The primary pathological abnormality appears to be loss of dopaminergic
neurons in the substantia nigra. In addition, eosinophilic cytoplasmic
inclusions termed Lewy bodies are present in nigral neurons. In cases of
post-encephalitic parkinsonism, neurofibrillary alterations are noted in
nigral neurons. The loss of these nigral cells leads to marked impairment
in the nigrostriatal pathway and a great diminution in the dopaminergic
synaptic input to the caudate and putamen. The enzymes of this incoming
pathway that synthesize dopamine are impaired [10]. Of importance is the
fact that no diminution is noted in the dopamine receptors within the
striatum. In fact, enhancement of receptor sensitivity may well be present
[11].
A number of pathological factors may impair nigrostriatal function and thus
give rise to secondary parkinsonism. These include infections and
post-infectious states; toxins such as manganese, carbon monoxide, or
carbon disulfide; drugs including neuroleptic compounds such as
phenothiazines, reserpine, and haloperidol; structural lesions such as
brain tumors, trauma, or syrinx or vascular disease; as well as metabolic
abnormalities such as hypoparathyroidism and basal ganglia calcification.
Alzheimer Disease
Alzheimer disease is a disorder of the later decades of life characterized
by dementia. In clinical terms, it consists of a diffuse deterioration of
mental function, primarily in thought and memory, and secondarily in
feeling and conduct. Alzheimer disease has been used to designate dementia
appearing before the age of 65 years. When the syndrome presents after
that age, the term senile dementia of the Alzheimer type is used. In fact,
it appears reasonable to consider both types as representing a single
syndrome. The true incidence of the disorder is unknown, although recent
data suggest that the incidence of all dementia in the U.S. population may
be over 100 cases per 100,000, with its prevalence being over 550 per
100,000 [12]; Alzheimer disease probably affects at least 30 to 50% of
patients with dementia, and in the United States there may be over one
million individuals with severe dementia and several million more with
mild to moderate dementia. It has been estimated that 1 out of every 6
persons over the age of 65 in the United States suffers from moderate
dementia, and a majority of patients in the nursing home populations are
affected with the disorder. The average age of onset is between 70 and 79
years, but without better information on the population at risk, a more
accurate statement is not presently possible [12]. As in ALS and
parkinsonism, the incidence of the syndrome clearly increases with
advancing age. A family history of Alzheimer disease is present in 5 to
10% of the patients.
At the present time, the clinical diagnosis of Alzheimer disease is one of
exclusion. Secondary causes of loss of memory and impaired cognitive
function may result from multiple infarcts, leading to so-called
multinfarct dementia, or from intracranial mass lesions such as subdural
hematomas, brain tumors, or granulomas. Central nervous system infections
of viral and bacterial origin, or even slow viral disorders such as
Jakob-Creutzfeldt disease, are part of the differential diagnosis.
Furthermore, metabolic disorders involving vitamin B.sub.12 metabolism,
thiamine or folate deficiency, thyroid dysfunction, hepatic and renal
failure, as well as drug toxicity, may present as dementia. Nevertheless,
when all these secondary causes, many of which are reversible, are
eliminated, cerebral atrophy of unknown cause or Alzheimer disease still
covers the largest number of patients. Elevations of aluminum content in
the brain have been implicated in the pathogenesis of the disorder but
appear to be secondary rather than primary [13, 14].
The pathological picture of Alzheimer disease has been well characterized
over the years. It consists of senile plaques, which result from
degeneration of nerve endings, and neurofibrillary tangles, which
represent an alteration in the cytoskeletal apparatus [15]. In addition,
intracellular cytoplasmic eosinophilic inclusions, termed Hirano bodies,
are present, primarily in the hippocampus. Granulovacuolar degeneration is
also noted. Senile plaques and neurofibrillary tangles in the brain are
part of the "normal" aging process. However, at any age, patients with
clinical Alzheimer disease appear to have much higher concentration of
these abnormalities than do normal individuals [16].
The most recent prominent discovery in Alzheimer disease is a deficiency of
the enzyme that synthesizes the neurotransmitter acetylcholine, namely,
choline acetyltransferase (CAT) [17]. This deficiency is most marked in
the cortex and hippocampus. Of note is the fact that acetylcholine
receptors in the brain are either unaffected or relatively less affected.
Thus, the defect in CAT reflects an alteration in the presynaptic
cholinergic neuron. The diminution in CAT correlates with the presence of
senile plaques: the greater the number of plaques, the lower the activity
of CAT. Enzymes synthesizing several other neurotransmitters, including
dopamine, norepinephrine, serotonin, and y-aminobutyric acid, as well as
levels of vasoactive intestinal peptide, are all relatively unaffected
compared to the loss of CAT activity. Somatostatin-like activity has
recently been reported to be decreased in the cerebral cortex [18].
The CAT activity found in the hippocampus appears to derive largely from
nerve terminals for which the cell of origin is in the septal nucleus. In
addition, almost 70% of CAT activity in the cortex appears to reside in
terminals with cell bodies located in the nucleus basalis of Meynert [19].
In rats, these cholinergic neurons lie intermingled with and beneath the
medial globus pallidus, whereas in primates comparable cells are found
exclusively outside the pallidum. In humans, the nucleus basalis of
Meynert is situated in the fibrous zone beneath the globus pallidus and is
a major component of the substantial innominata [20]. Thus, the
cholinergic input to hippocampus and cortex may derive from a group of
cells extending from the septal nuclei to constituents of the substantia
innominata and may well be impaired in Alzheimer disease [20].
The following references are relevant to the invention
1. Munsat T. L., Bradley W. G.: Amyotrophic lateral sclerosis. In Tyler H.
R., Dawson D. M. (eds): Current Neurology, vol 2. Boston, Houghton
Mifflin, 1979
2. Juergens S. M., Kurland L. T., Okazaki H., Mulder D. W.: ALS in
Rochester, Minn., 1925-1977. Neurology (NY) 30:463-470, 1980.
3. Engel W. K.: Motor neuron disorders. In Goldensohn E. S., Appel S. H.
(eds): Scientific Approaches to Clinical Neurology. Philadelphia, Lea &
Febiger, 1977, pp 1322-1346
4. Dyck P. J., Stevens J. C., Mulder D. W., et al: Frequency of nerve fiber
degeneration of peripheral motor and sensory neurons in amyotrophic
lateral sclerosis: morphometry of deep and superficial peroneal nerve.
Neurology (Minneap) 25:781-785, 1975
5. Chou S. M.: Pathognomy of intraneuronal inclusion in ALS. In Tsubaki T,
Toyokura Y (eds): Amyotrophic Lateral Sclerosis. Tokyo, University of
Tokyo Press, 1979, pp 135-176
6. Bradley W. G., Kelemen J., Adelman L. S., et al: The absence of
dying-back in the phrenic nerve of amyotrophic lateral sclerosis (ALS).
Neurology (NY) 30:409, 1980
7. Carpenter S.: Proximal axonal enlargement in motor neuron disease.
Neurology (Minneap) 18:841-851, 1968
8. Griffin J. W., Hoffman P. N., Clark A. W., Carrol P. T., Price D. L.:
Slow axonal transport of neurofilament proteins: impairment of beta,
beta-iminodipropionitrile administration. Science 202:633-635, 1978
9. Marttila R. J., Rinne U. K.: Changing epidemiology of Parkinson's
disease: predicted effects of levodopa treatment. Acta Neurol Scan
59:80-87, 1979
10. Calne D. B., Kebabian J. Silbergeld E., et al: Advances in the
neuropharmacology of parkinsonism. Ann Intern Med 90:219-229, 1979.
11. Burke R. E., Fahn S.: Movement Disorders. In Appel S. H. (ed): Current
Neurology. New York, Wiley, 1981, vol 3, pp 92-137
12. Schoenberg B.: personal communication, 1981
13. Crapper D. R., Quittrat S., Krishnau S. S., Dalton A. J., DeBon U.:
Intranuclear aluminum content in Alzheimer's disease, dialysis
encephalopathy and experimental aluminum encephalopathy. Acta Neuropathol
(Berl) 50:19-24, 1980
14. Perd D. P., Brody A. R.: Alzheimer's disease: x-ray spectrometric
evidence of aluminum accumulation in neurofibrillary tangle-bearing
neurons. Science 208:297-299, 1980
15. Terry R. D. Davies P.: Dementia of the Alzheimer type. Annu Rev
Neurosci 3:77-95, 1980
16. Blessed G., Tomlinson B. E., Roth M.: The association between
quantitative measures of dementia and of senile change in the cerebral
grey matter of elderly subjects. Br. J. Psychiatry 114:797-811, 1968
17. Davies P., Maloney A. J. F.: Selective loss of central cholinergic
neurons in Alzheimer's disease. Lancet 2:1403, 1976
18. Davies P., Katzman, R., Terry R. D.: Reduced somatostatin-like
immunoreactivity in cerebral cortex from cases of Alzheimer's disease and
Alzheimer senile dementia. Nature 288:279-280, 1980
19. Johnston M. V., McKinney M. Coyle J. F.: Evidence for a cholinergic
projection to neocortex from neurons in basal forebrain. Proc Natl Acad
Sci U.S.A. 76:5392-5396, 1979
20. Whitehouse P. J., Price D. L., Clark A. W., Coyle J. T., DeLong M. R.:
Alzheimer disease: evidence for selective loss of cholinergic neurons in
the nucleus basalis. Ann Neurol 10:122-126, 1981
The following additional references are also relevant to the invention:
Bottenstein J. E., Sato G. H.: Growth of a rat neuroblastoma cell line in
serum-free supplemented media. Proc Natl Acad Sci U.S.A. 76:514-517, 1979
Bradshaw R. A.: Nerve growth factor. Annu Rev Biochem 47:191-216, 1978
Brown M. C., Holland R. L., Hopkins W. G.: Motor nerve sprouting. Annu Rev
Neurosci 4:17-42, 1981
Cohen J. Levi-Montalcini R.: A nerve growth-stimulating factor isolated
from snake venom. Proc Natl Acad Sci U.S.A. 42:571-574, 1956
Davies P.: Loss of choline acetyltransferase activity in normal aging and
in senile dementia. Adv Exp Med Biol 113:251-257, 1978
Finch C. E.: Catecholamine metabolism in the brains of aging male mice.
Brain Res 52:261-276, 1973
Fonnum F.: Radiochemical micro assays for the determination of choline
acetyltransferase and acetycholinesterase activities. Biochem J.
115:465-472, 1969
Giller E. L., Neale J. H., Bullock P. N., Schrier B. K., Nelson P. G.:
Choline acetyltransferase activity of spinal cord cell cultures increased
by co-culture with muscle and by muscle-conditioned medium. J. Cell Biol
74:16-29, 1977
Hemmendinger L. M., Garber B. B., Hoffman P. C., Heller A.: Target
neuron-specific process formation by embryonic mesencephalic dopamine
neurons in vitro. Proc Natl Acad Sci U.S.A. 78:1264-1268, 1981
Hollyday M., Hamburger V.: Reduction of the naturally occurring motor
neuron loss by enlargement of the periphery. J. Comp Neurol 170:311-320,
1976
Hudson A. J.: Amyotrophic lateral sclerosis and its association with
dementia, parkinsonism and other neurological disorders: a review. Brain
104:217-247, 1980
Johnson D. A., Pilar G.: The release of acetylcholine from post-ganglionic
cell bodies in response to depolarization. J. Physiol (Lond) 299:605-619,
1980
Mobley W. C., Server A. C., Ishii D. N., Riopelle R. J., Shooter E. M.:
Nerve growth factor. N Engl J Med 297:1096-1104, 1977
Pestronk A., Drachman D. B., Griffin J. W.: Effects of aging on nerve
sprouting and regeneration. Exp Neurol 70:65-82, 1980
Pittman R. W., Oppenheim R. W.: Neuromuscular blockage increases
motoneurone arrival during normal cell death in the chick embryo. Nature
271:364-366, 1978
Prochiantz A., DiPorzio U., Kato A., Berger B., Glowinski J.: In vitro
maturation of mesencephalic dopaminergic neurons from mouse embryos is
enhanced in presence of their striatal target cells. Proc Natl Acad Sci
U.S.A. 76:5387-5391, 1979
Reed D. M., Torres J. M., Brody J. A.: Amyotrophic lateral sclerosis and
parkinsonian-dementia on Guam, 1945-1972. Am J Epidemiol 101:302-310, 1975
Smith R. G., Appel S. H.: Evidence for a skeletal muscle protein that
enhances neuron survival, neurite extension, and acetylcholine (ACh)
synthesis. Soc Neurosci Abstr 11:144, 1981
U.S. Pat. No. 4,294,818 discloses a diagnostic method for multiple
sclerosis comprised of antibody preparations reactive with antigenic
substances associated with lymphocytes.
U.S. Pat. No. 3,864,481 discloses a synthetic amino acid for suppression
and diagnosis of multiple sclerosis.
U.S. Pat. Nos. 3,961,894; 4,046,870; and 4,225,576 disclose assay
techniques for detecting hormones in the body.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that ALS, parkinsonism,
and Alzheimer disease result from lack of a neurotrophic factor specific
for a particular neuronal network or system which is elaborated or stored
in the synaptic target of the affected neurons and exerts a specific
effect by acting in a retrograde fashion. Diagnosis and treatment are
based on neurotrophic factors which are extracted and tested in three
different systems: in ALS the muscle factor which enhances motor neuron
survival, growth and development; in parkinsonism the striatal factor
which enhances substantial nigra survival, growth and development; and in
Alzheimer disease the hippocampus factor which enhances septal neuron
survival, growth and development. These neurotrophic factors are
extracted, purified, and assayed. Diagnosis is accomplished for ALS by
assaying motor neurotrophic factors from muscle in the motor system, for
parkinsonism by assaying dopamine neurotrophic factors from striatum in
the nigrostriatal system, and for Alzheimer disease the cholinergic
neurotrophic factor released from the cortex and hippocampus. In case of
deficiencies, neurotrophic factors specific to the particular neuronal
network or system are injected into patients with ALS, parkinsonism and
Alzheimer disease.
Accordingly, it is an object of the present invention to provide effective
diagnosis and treatment of ALS, parkinsonism, and Alzheimer disease by
neurotrophic factors.
It is a further object of the present invention to diagnose parkinsonism by
determining or assaying the dopamine neurotrophic factor from striatum
specific for the nigrostriatal system.
It is a further object of the present invention to treat parkinsonism by
injecting neurotrophic factors specific to the nigrostriatal system.
A further object of the present invention is the extraction and
purification of neurotrophic factors specific for the motor system, for
the nigrostriatal system, and for cholinergic neurons of the nucleus
basalis and septal nucleus.
Other and further objects, features and advantages of the invention are set
forth throughout the specification and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of gel filtration chromatography of the striatal
extract run on a Biogel P4 column as well as a dose response of the major
fractions of the Biogel P4 column on the stimulation of high affinity
dopamine and GABA uptake.
DESCRIPTION OF PRESENTLY PREFERRED EMBODIMENTS
From the foregoing, all three diseases can be seen to represent disorders
of specific neuronal networks; that is, the motor neuronal system, the
nigrostriatal dopaminergic neuronal system and the cholinergic neuronal
system. All reflect changes in a presynaptic neuronal input with secondary
alterations of the target tissue. ALS represents pathological change in
Betz cells, cranial motor neurons, and anterior horn cells; parkinsonism,
in substantial nigra dopaminergic neurons; and Alzheimer disease, in the
cholinergic input from nucleus basalis and septal neurons to cortex and
hippocampus, respectively.
The role of neurotrophic hormones of the present invention is a
modification of the notion of intrinsic aging of selected neurons; that
is, the presence of specific extrinsic factors influence the maintenance
and survival of neurons. In each disease, the system degeneration is due
to diminished availability of a specific neurotrophic hormone normally
released by the post-synaptic cell, taken up by the presynaptic terminal,
and exerting its effect by retrograde transport up the presynaptic axon to
the soma and nucleus.
For each of these three neuronal systems, neurotrophic factors can be
demonstrated in vitro which enhance neuron survival, promotes neurite
extension, and increase the activity of the neurotransmitter synthetic
enzymes in the innervating cell. The same factors responsible for survival
of neurons in vitro may also be responsible for survival of neurons in
vivo. Similar or even the same factors may also be responsible for
maintenance of neurons throughout the life cycle in vivo, and may decrease
as a normal function of aging.
Thus, a primary manifestation of ALS, Parkinson disease, or Alzheimer
disease is failure of the target tissue to supply the necessary
neurotrophic hormone. Marked pathological change in the tissue need not be
present. Impaired synthesis or release (or both) of the relevant hormone
would represent the sine qua non of disease. For example, in the lower
motor neuron syndromes of ALS, failure of muscle cells to release the
appropriate motor neurotrophic hormone would result in failure of anterior
horn cells. The pathological picture would be one of gradual cessation of
anterior horn cell function with chromatolysis and of altered nuclear
function with minimal evidence of "dying back." Similarly, impairment of
Betz cells would result from decreased release of neurotrophic hormone
from target neurons. A more precise statement is not possible for the
upper motor neuron syndrome since the synaptic target of the descending
Betz cell axon is not known with certainty in humans.
In parkinsonism, the neurotrophic failure would be characterized by
inability of striatal cells to provide the required dopamine neurotrophic
hormone. In Alzheimer disease, the failure would be in hippocampus and
cortical cells to supply the relevant cholinergic neurotrophic hormone.
Thus, in each system, the lack of an appropriate hormone released from
post-synaptic cells impairs the viability of the presynaptic cells.
Anterior horn cells, Betz cells, substantia nigra cells, and septal and
basal nuclei undergo gradual deterioration.
Thus, motor neurotrophic hormones are released from muscle and are specific
for the motor system, dopamine neurotrophic hormones are released from the
striatum and are specific for the nigrostriatal dopaminergic system, and
cholinergic neurotrophic hormones are released from the cortex and
hippocampus and are specific for cholinergic neurons of the nucleus
basalis and septal nucleus. With the availability of tissue culture, the
presence, deficiency, or absence of specific neurotrophic hormones can be
assessed in ALS, parkinsonism, and Alzheimer disease readily and easily.
The present invention discloses a method to prepare a composition effective
in treating Parkinson's disease comprising:
(a) extracting factors from the caudate-putamen tissue of a normal mammal;
and
(b) assaying the caudate-putamen extract for trophic effects on
dopaminergic neurons obtained from substantia nigra.
The method of the present invention further comprises the isolation of
three factors with the following molecular weight ranges: 1800-2600
daltons (Factor I); 1500-1800 daltons (Factor II); and 1000-1600 daltons
(Factor III). The molecular weight ranges of the factors described herein
have been determined in solution by gel filtration chromatography and
thus, have been given in ranges since the specific molecular weight may
vary according to the procedure used to isolate each factor.
The tissue extract used in the present invention is derived from either
striatum tissue from rat or caudate and putamen tissue from bovine
sources. The tissue sample is derived from those regions of the brain
innervated by dopaminergic neurons of the substantia nigra, such as the
striatum and the caudate-putamen, which are rich in dopaminergic
neurotrophic factors. Significant amounts of dopaminergic neurotrophic
activity can be obtained in extracts of the striatum, the
hippocampus-entorhinal cortex-amygdaloid cortex area and the cerebral
cortex. The precise region of the brain used is dependent upon the type of
mammal used as the source of tissue. For example, in the rat, the
preferred tissue is striatum whereas preferred bovine midbrain tissue are
the caudate and putamen. Both the striatal tissue and the caudate-putamen
tissues are regions of the brain innervated by dopaminergic neurons of the
substantia nigra.
By "trophic effects" it is meant that the extracted neurotrophic factors
have selective effects on specific neural elements, said effects
contributing to the survival, growth, maturation, differentiation, and
regeneration of neurons present in the nervous tissue.
Biological assays for neurotrophic factors active on either dopaminergic
neurons or GABAergic (gamma alpha aminobutyric acid containing) neurons
are used as one means of demonstrating the trophic effects possessed by
the present neurotrophic factors. Dopaminergic activity is usually defined
as the ability to stimulate specific high affinity .sup.3 H-dopamine
uptake by dopaminergic neurons. In accord, GABAergic activity is the
ability to stimulate high affinity .sup.3 H-GABA uptake by GABAergic
neurons. Both of these assays were conducted because the substantia nigra
in the mesencephalon contains dopaminergic and GABAergic neuronal
populations.
Very generally, the trophic factors which exhibit trophic effects on
dopaminergic neurons can be isolated from caudate and putamen tissues from
human or bovine sources or striatal tissue from rat. Thus, the source of
tissue can be taken from a variety of normal mammals. The use of the term
"striatal" and "caudete-putamen" herein refers to the regions of the brain
innervated by dopaminergic neurons of the substantia nigra, regardless of
tissue source.
The neurotrophic factors described herein can be isolated using a variety
of conventional and well-known extraction and purification procedures.
Extraction procedures include homogenizing the tissue in an aqueous
solution using a blender or laboratory homogenizer. The aqueous solution
is usually buffered to physiological salt and pH and may contain one or
more protease inhibitors including one or more reducing agents and one or
more chelating agents. The particular buffers, reducing agents, and
protease inhibitors used in the extraction step are not critical to
practice the present invention; thus, the following reagents are merely
illustrative of the reagents which might be utilized. For example, the
aqueous solution may include phosphate-buffered saline solution (PBS),
citrate-phosphate buffer; protease inhibitors may include trypsin
inhibitor, phenylmethylsulfonyl fluoride (PMSF), pepstatin A, and
leupeptin; chelating agents may include EDTA, EGTA and the like; and
reducing agents may include .beta.-mercaptoethanol, dithiothreitol, and
the like. The factors described in these examples are stable at moderate
acid and basic pHs. Therefore, extraction could be in acidic or basic
aqueous solutions such as acetic acid or ethanolamine solutions. A
preferred embodiment of the present invention utilizes PBS, pH 7.4,
supplemented with soybean trypsin inhibitor and .beta.-mercaptoethanol to
extract the dopamine neurotrophic factors. Tissue extracts prepared by
this tissue homogenization are then clarified by centrifugation.
The recovery of Factor I is particularly dependent on the freshness of the
tissue used in the extraction process. In general, it is best to use
tissue from 6-12 month old calves. Thus, Factor I may be recovered in
greater yield if the tissue is from a 6 month old calf and is removed from
the calf less than two hours prior to the extraction protocol. In
addition, if a trypsin inhibitor and .beta.-mercaptoethanol are not
present in the buffer used for extraction, recovery of Factor I is greatly
reduced.
Several fractionation procedures which can be used singly or in combination
to increase purity of a composition are well known in the art. These
include: size fractionation using molecular sieve (or gel filtration)
chromatography, ion exchange chromatography under suitable conditions;
affinity chromatography using for example, antibodies directed to the
biologically active form of the neurotrophic factor; absorption
chromatography using non-specific supports, such as hydroxyapatite,
silica, alumina, and so forth; and also gel supported electrophresis. A
detailed description of the procedures used to purify the present
neurotrophic factors is described in the examples below.
Since the factors described herein are relatively small (<3,000 daltons),
undesired proteins and cellular debris can be removed by ultrafiltration
of the extract through an appropriate filter, for example, an Amicon YM10
filter. Alternatively, and preferably, the extracted dopamine neurotrophic
factors may be directly purified by gel filtration chromatography using an
appropriate matrix to resolve proteins having molecular weights less than
3,000 daltons. The matrix, such as a Biogel P4 column, is preequilibrated
or equilibrated with an appropriate buffer such as 25 mM ammonium
bicarbonate, pH 6.5, which is also useful to elute the neurotrophic
factors.
Dopamine uptake stimulating activity is separated into three peaks of
activity under gel filtration chromatography. The peak with the highest
molecular weight (.about.1800-2600 daltons) was designated Factor I;
Factor II (.about.1500-1800 daltons) elutes consistently just in front of
Factor III (.about.1000-1600 daltons).
The factors in these three active peaks were assayed for enhancement and
differentiation of dopaminergic neurons. Mesencephalon tissue is obtained
from mammalian species for the assay. It is preferred to use mesencephalic
cells from E14 rat embryos either for dissociated cell culture or tissue
explant culture.
Several different assays may be performed to determine the nature and
specificity of the effects of the factors on the dopaminergic neurons in
these cultures. These include effects on survival, cell growth, neuronal
process outgrowth, and general stimulation of metabolic functions
including those specifically related to neuronal type. For example,
enhancement of levels of enzymes such as tyrosine hydroxylase which are
involved in this transmitter synthesis may be determined. The preferred
method of assay is to incubate cultures of rat dopaminergic neurons with
or without each of the neurotrophic factors for several days after which
specific effects on the dopaminergic neurons are determined. Usually, high
affinity dopamine uptake is measured by incubating treated cultures with
.sup.3 H-dopamine for 80 min at 22.degree. C. Appropriate controls allow
determination of the ability of each factor to specifically stimulate high
affinity dopamine uptake. Dopaminergic neurons can be identified in
cultures by staining for dopamine by glyoxylic acid-induced catecholamine
histofluorescence.
Besides dopaminergic neurons, mesencephalon cultures contain large amounts
of GABAergic (gamma-aminobutyric acid containing) neurons. Therefore, one
way of determining neurotrophic specificity for dopaminergic neurons is by
the assaying for the effect of each factor on high affinity GABA uptake
which is carried out in a similar fashion using .sup.3
H-.gamma.-aminobutyric acid. Factors (e.g., Factor II) which stimulate
GABA uptake are not dopaminergic specific, while only those factors (e.g.,
Factors I and III) which stimulate dopamine uptake and do not stimulate
GABA uptake are dopaminergic specific. The lack of tight specificity of
Factor II for dopaminergic neurons does not preclude its utility, since
trophic factors with broad spectrum of action may also be effective in
treating Parkinson's disease.
The present neurotrophic factors may be further purified by ion-exchange
chromatography. The fractions corresponding to the Factor III isolate can
be purified away from any contaminating Factor II component by
anion-exchange high performance liquid chromatography (HPLC). The Factor
III eluate is dissolved in a low salt buffer and adjusted to pH
.about.7.0. Usually, a low salt phosphate buffer such as 10 mM NaH.sub.2
PO.sub.4 -Na.sub.2 HPO.sub.4 is used. This solution is adsorbed to an
anion exchange HPLC column such as DEAE-5-PW-equilibrated with a similar
buffer. This column is eluted isocratically for a time and then with a
linear gradient of NaCl from 0 to 250 mM. The Factor III neurotrophic
activity elutes between 50-60 mM NaCl whereas the Factor II neurotrophic
activity elutes around 140 mM NaCl.
The Factor I activity can be purified by cation-exchange HPLC by dissolving
the Factor I eluate in a low salt phosphate buffer and adjusting the pH to
4.5. The resulting solution is applied to a cation exchange (e.g.,
SP-5-PW) HPLC column preequilibrated or equilibrated with 10 mM phosphate
buffer, pH 4.5. After washing thoroughly, the desired factor is
isocratically eluted with the pH 4.5 phosphate buffer for five minutes and
then eluted in a linear gradient at 0 to 250 mM NaCl. The Factor I
neurotrophic activity elutes between 50-60 mM NaCl.
Each of the factors isolated above may be subjected to further purification
protocols and assayed for dopaminergic activity as described hereinabove.
However, the individual factors isolated according to the procedures
described in the following examples have been characterized sufficiently
to determine their ability to enhance process growth and specific dopamine
uptake in mesencephalon cultures.
Extracts of the striatum of the mammalian brain influence the survival,
development and differentiation of substantia nigral dopaminergic neurons.
The importance of this isolation and purification is that such a factor
may not only play a role in the development of dopaminergic innervation
during development, but it may also play a role in maintenance of such
innervation. Thus, in diseases such as Parkinson's disorder in which
substantia nigra cells are lost, such a dopaminergic neurotrophic factor
may be deficient. Replacement of this particular neurotrophic hormone may
have salutary effects on the clinical syndrome of Parkinson's disease.
Accordingly, the invention further comprises a method of diagnosing the
presence of Parkinson's disease in a subject by detecting a deficiency of
specific neurotrophic functional effects in said subject as compared to
normal controls, wherein said deficiency is detected by the method which
comprises:
(a) extracting a component from the caudate-putamen tissue of said subject;
(b) assaying said extract for neurotrophic activity with respect to the
neuronal system normally associated with said caudate-putamen; and
(c) comparing said neurotrophic activity to activity exhibited in the same
assay by similar extracts from caudate-putamen tissue of controls.
The component may be prepared as described in the following examples and
assayed for dopaminergic activity in the nigrostriatal pathway. If a
deficiency in dopaminergic neurotrophic hormone levels is identified,
treatment may be accomplished by injecting the present factors.
The factors prepared as described herein are suitable for parenteral
administration to humans or other mammals in therapeutically effective
amounts (e.g., amounts which eliminate or reduce the patient's
pathological condition) to provide therapy for Parkinson's disease.
Additionally, the factors may be useful in transplant therapy as they will
have a supportive effect on survival and maintenance of dopaminergic
tissues or cells transplanted to the Parkinsonian brain in order to
replace the lost dopaminergic function.
The formulations of this invention are useful for parenteral
administration, for example, intravenous, subcutaneous, intramuscular,
intraorbital, opthalmic, intracapsular, intraspinal, intrasternal,
topical, intranasal aerosol, scarification, and also, for oral
administration. The preferred route of administration would be by
intranasal aerosol.
The concentration of neurotrophic factor in a therapeutic composition will
vary depending on a number of factors, including the dosage of the drug to
be administered, and the chemical characteristics, e.g., hydrophobicity,
of the factor. Generally, the factor is provided in an aqueous
physiological buffer solution containing about 0.1 to 10% w/v factor.
Other adjuvants include glycocholate, deoxycholate and sodium
tauroglycocholate.
The invention is further described by the following examples. These
examples are not intended to limit the invention in any manner.
EXAMPLE 1
Purification of Dopaminergic Factors
A. Extraction
About 30 g of 6 month old calf caudate-putamen tissue on ice was
homogenized in five volumes (wt/vol) of PBS (1/3 of normal PBS, pH 7.0) in
the presence of 0.1 mg/ml soybean trypsin inhibitor and 0.5 mM
beta-mercaptoethanol and allowed to stand for 30 min on ice. The extract
was prepared by centrifugation of the PBS homogenate at 100,000 g for 90
min. The extraction was repeated one more time under the same conditions
and the resulting supernatant filtered through a 2 micron filter,
lyophilized, and assayed for trophic activity as described in Example 2.
B. Gel Filtration Chromatography
The lyophilized PBS extract was dissolved in 8 ml distilled water
containing 0.5 mM beta-mercaptoethanol and 0.1 mg/ml trypsin inhibitor.
This suspension was applied to a Biogel P4 (BioRad) molecular sieving
column preequilibrated with 25 mM ammonium bicarbonate buffer, pH 6.5.
Fractions were collected and protein concentrations were assessed by a
modification of the method of Lowry (Schacterle and Pollack, Anal Biochem
(1973) 51: 654-655). The collected fractions were also assayed for
stimulation of high affinity dopamine uptake and neuronal high affinity
GABA uptake as described below. Three peaks of stimulatory activity of
high affinity of dopamine uptake were observed which correspond to the
following approximate molecular weights: 1800-2600 daltons (Factor I);
1500-1800 daltons (Factor II); and 1000-1600 daltons (Factor III). The
peak with the highest molecular weight, Factor I, appears to be quite
sensitive to protease activity. The presence of this peak is variable and
is dependent on the age and freshness of calf brain tissue.
The stimulatory effects of these three active peaks on high affinity
dopamine uptake and neuronal high affinity GABA uptake were compared and
the results are shown in FIG. 1. Factors I and III stimulate high neuronal
high affinity dopamine uptake in dose-dependent manner, but do not
stimulate neuronal high affinity GABA uptake. Factor II, in contrast,
stimulates both high affinity dopamine uptake and neuronal high affinity
GABA uptake. Therefore, Factor I and Factor III appear to be specific for
dopaminergic neurons while Factor II is less specific being neurotrophic
for both dopaminergic and GABAergic neurons in dissociated mesencephalon
cultures.
C. Anion-Exchange HPLC Chromatography of Factor III
The Biogel P4-Factor III fractions, including some overlapped areas of
Factor II, were lyophilized and dissolved in 2 ml phosphate buffer (10 mM
NaH.sub.2 PO.sub.4 -Na.sub.2 HPO.sub.4, pH 7.0). Factor III was applied to
a preequilibrated (with the same phosphate buffer) DEAE-PW-HPLC column
(7.5 mm.times.7.5 cm, Waters Associates). The column was eluted
isocratically for 20 min, and then with a linear gradient of NaCl from 0
to 250 mM in the phosphate buffer at a flow rate of 0.5 ml/min. A peak of
dopaminergic stimulating activity eluted at 50-60 mM NaCl. As shown in
FIG. 1, this fraction stimulated high affinity dopamine uptake, but not
neuronal high affinity GABA uptake. A second peak of activity eluted at
140 mM NaCl. This peak, however, stimulated both dopamine and GABA uptake
and is therefore Factor II. Thus, dopaminergic Factor III can be
effectively separated from Factor II by DEAE chromatography resulting in a
purification of approximately 5,000-6,000 fold purification over the
activity in the crude extract. Correspondingly, Factor II may be isolated
by pooling fractions from the P4 chromatography step which contain Factor
II plus some overlap of Factor III and similarly subjecting the pooled
fractions to anion-exchange chromatography. A summary of purification
results for Factor III is provided in Table 1 below.
TABLE 1
__________________________________________________________________________
PURIFICATION OF FACTOR III
ACTIVITY
PROTEIN
SA
FRACTION
(mg) (U/mg)
UNITS Ka
__________________________________________________________________________
100,000 xg
728 .+-. 42
25 18360 .+-. 2440
153 .mu.g/ml .+-. 15 .times. 10.sup.3
SPN
P4 (III)
24 .+-. 5
508 12200 .+-. 1700
5 .mu.g/ml .+-. 0.8 .times. 10.sup.3
DEAE (50- 30 ng/ml .+-. 12
60 mM Pool)
__________________________________________________________________________
D. Cation-Exchange HPLC Chromatography of Factor I
The Biogel P4-Factor I fractions were dissolved in 1 ml phosphate buffer
(10 mM NaH.sub.2 PO.sub.4 -Na.sub.2 HPO.sub.4, pH 4.5) and applied to a
preequilibrated (with the same phosphate buffer) SP-5-PW HPLC column (7.7
mm.times.7.5 cm, Waters Associates). Bound material was isocratically
eluted with the pH 4.5 phosphate buffer for 5 min, and then eluted in an
increasing linear gradient of NaCl from 0 to 250 mM. A peak of
dopaminergic stimulating activity eluted at 50-60 mM NaCl. This
dopaminergic factor, Factor I, was purified 6,500 fold over the activity
in the crude extract at the stage of SP-5-PW HPLC.
EXAMPLE 2
Assay Methods
A. Tissue Cultures
Explant and dissociation cultures of the substantia nigra were obtained
from 14-day-old rat embryos. At this stage of development, the
dopaminergic neurons in the mesencephalon are predominantly postmitotic,
but have not yet innervated their striatal target. Synthesis of dopamine
was detected by histofluorescence using the procedure by Specht et al (J
Comp Neurol (1981) 199: 233-253; ibid 255-276). For explant cultures, the
mesencephalon was dissected out and the ventral region fragment was
dissected into 0.3 to 0.4 mm pieces in the culture medium. Thirty to forty
pieces were cultured on poly-L-lysine (Sigma, 3,000,000 M.W.
polymerization) coated 35 mm Falcon tissue culture plates in a standard
culture medium consisting of 50% high glucose DMEM-50% F12 supplemented
with insulin (5 .mu.g/ml), transferrin (100 .mu.g/ml), progesterone (20
nM), putrescine (100 .mu.M), selenium (30 nM), glutamine (4 mM),
gentamycin (50 .mu.g/ml) and HEPES (5 mM) (Bottenstein and Sato, Proc.
Natl Acad Sci U.S.A. (1979) 76: 514-518) without serum and maintained at
37.degree. C. in a 95% air-5% CO.sub.2 humidified incubator. Dissociation
cultures were prepared by triturating mesencephalic cells in the presence
of DNase (1 mg/ml, Sigma) and trypsin (0.2 mg/ml, Sigma) (McCarthy and de
Vellis, J Cell Biol (1980) 85: 890-902). After washing twice with culture
medium containing 4% heat inactivated horse serum, cells were plated at
100,000 cells/well on the poly-L-lysine coated Falcon 96 multiwell plates
in the same culture medium. Six hours after plating, the soluble factors
were applied onto the medium with cytosine arabinoside (2 .mu.M) and the
cells were cultured for three days total in a 95% air-5% CO.sub.2
humidified incubator.
B. Dopaminergic Activity
Assays of high affinity .sup.3 H-dopamine uptake were performed by the
method reported by Prochiantz et al, Nature (1981) 293: 570-572. Cells
were incubated at 22.degree. C. for 80 min with 50 nM H-dopamine (New
England Nuclear, 20-30 Ci/mmole) in 150 ul of PBS containing 6 mg/ml
glucose, 1 mg/ml BSA, and 40 ug/ml ascorbic acid, pH 7.0 in the presence
of pargyline (100 .mu.M), an inhibitor of monoamine oxidase. The assays
were terminated by washing cells four times with ice-cold PBS without
CaCl.sub.2 and MgCl.sub.2. Cells were lysed by addition of 150 ml of 0.5N
NaOH for 2 hours at room temperature. Incorporated .sup.3 H-dopamine was
measured by a liquid scintillation counter. .sup.3 H-dopamine uptake by
dopaminergic neurons was verified by specific inhibition by benztropine
and desmethylimpramine at 1 .mu.M (Prochiantz et al, 1981, supra). In the
presence of both inhibitors the high affinity dopamine uptake in these
experiments was inhibited by 87%. To determine the non-specific dopamine
uptake, dissociated cells, enriched in glia, were prepared from 1-day-old
rat cerebral cortex, and cultured at the similar cell density. The
population was 98% glia by GFAP staining. Dopamine uptake in this culture
was less than 3% of the PBS control of mesencephalon cultures.
C. GABAergic Activity
The major neuronal constituents in mesencephalon cultures are dopaminergic
and GABAergic neurons. The striatal factors were, therefore, tested for
their ability to enhance high affinity GABA uptake. Mesencephalic cells
were incubated at 22.degree. C. for 80 minutes with 100 nM .sup.3
H-.gamma.-aminobutyric acid (GABA) (ICN, 75 Ci/mmole) in PBS, pH 7.4, in
the presence of .beta.-alanine (2 mM), an inhibitor of glial low affinity
GABA uptake. High affinity .sup.3 H-GABA uptake specific for GABAergic
neurons was verified with respect to Factor II, with the inhibitor L-2,
4-diaminobutyric acid (1 mM) (Pastuszko et al, Proc Natl Acad Sci U.S.A.
(1981) 78: 1242-1244).
D. Catecholamine Histofluorescence
Dissociated cultured mesencephalon cells were analyzed by catecholamine
fluorescence using a glyoxylic acid technique (Sumners et al, Brain Res
(1983) 264: 267-275). Cells grown on glass multichamber slides were rinsed
in ice-cold PBS, pH 7.4, and immediately placed in a 1% buffered glyoxylic
acid solution (1% glyoxylic acid, 0.1M phosphate, pH was adjusted to pH
7.4 with NaOH 4.degree. C.) for 5 minutes. Excess glyoxylic acid solution
was removed. Slides were dried for 5 minutes under warm air, and then
heated for 10 minutes at 95.degree. C. Fluorescence was viewed under a
Nikon fluorescence microscope with V filter block (IF 399-425 interference
excitation filter and 470 barrier filter). Control cells were treated with
cold PBS for the same period. To intensify dopamine histofluorescence,
cells were pretreated 5 hours with 100 .mu.M L-Dopa in 100 .mu.g/ml
ascorbic acid and 100 .mu.M pargyline. Non-dopaminergic neurons and glial
cells did not show any specific glyoxylic acid induced-catecholamine
fluorescence under these conditions.
Top